title: Construction of vectors for inducible and constitutive gene expression in Lactobacillus.
abstract: Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.
authors.affiliation: Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, USA.
authors.forename: Tri
authors.initials: T
authors.lastname: Duong
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Miller
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.forename: M Andrea
authors.initials: MA
authors.lastname: Azcarate-Peril
authors.forename: Todd R
authors.initials: TR
authors.lastname: Klaenhammer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D007778:Lactobacillus / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins